Preparation method and application of pre-F fusion protein of recombinant RSV

Abstract

The invention discloses a preparation method and application of a Pre-F protein of a respiratory syncytial virus, and a cell culture solution with high protein expression yield and high protein purity is obtained by optimizing process parameters in a cell culture process, such as a basic culture medium, a fed-batch culture medium, inoculation density, temperature, time and the like. And a good basis is provided for preparation of the respiratory syncytial virus vaccine.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and relates to a preparation method and application of a recombinant RSV Pre-F fusion protein. Background Art

[0002] RSV is the abbreviation of Respiratory Syncytial Virus (RSV). It was first discovered in 1955 and belongs to the family Paramyxoviridae, subfamily Pneumovirinae, genus Pneumovirus. It can be divided into two subtypes, A and B, based on the sequence of the G protein. RSV is a non-segmented negative-strand RNA virus with a genome length of 15.2kb. It has 10 genes and encodes a total of 11 proteins, including non-structural proteins (NS1, NS2), nucleoprotein (N), phosphoprotein (P), matrix protein (M), RNA-dependent RNA polymerase (L), transcription elongation factor (M2-1), regulatory factor (M2-2) and 3 envelope glycoproteins (adhesion protein (G), fusion protein (F) and small hydrophobic protein (SH)).

[0003] RSV is a viral pathogen that causes respiratory tract infection (RTI). Infection mainly causes symptoms of lower respiratory tract infection, among which patients with severe symptoms account for a considerable proportion (such as bronchiolitis and pneumonia), requiring hospitalization and having a high mortality rate. RSV can be transmitted through contact between people, or inhaled through coughing or sneezing, or acquired through contact with pollutants. It mainly infects epithelial cells of the nasal cavity and large and small airways of the lungs, and may also infect alveolar macrophages and other types of cells in the lungs, causing cells to fuse together to form syncytia.

[0004] The RSV antigenic protein Pre-F protein is the pre-fusion conformation of RSV F protein and is mainly expressed in host cells through biological methods. However, the current protein yield is not high. It is urgent to explore the optimal preparation process to obtain a higher concentration of Pre-F protein and thus obtain a vaccine with better immunity. Summary of the invention

[0005] In one aspect of the present invention, a method for culturing cells is provided, wherein the cells express Pre-F protein, and the method comprises culturing the cells using B602S, B600S or AltC as a basic culture medium.

[0006] Preferably, the basal medium comprises AltC.

[0007] Preferably, the culture method comprises using F600aS / F600bS, F600aS / ZY-S001, F601aS / F600bS or AltairCHO Feed / CDFS36 as feed medium;

[0008] More preferably, the feed medium comprises AltairCHO Feed / CDFS36.

[0009] More preferably, the feed volume ratio of the feed medium AltairCHO Feed / CDFS36 is (8-12) / 1, which can be any data or range within the above range, such as 8 / 1, 9 / 1, 10 / 1, 11 / 1, 12 / 1. In a specific embodiment, the feed volume ratio is 10 / 1.

[0010] More preferably, the single AltairCHO Feed / CDFS36 feed concentration is (1.5%-6%) / (0.15%-0.6%), which can be any data or range within the above range, for example, 1.5% / 0.6%, 2.0% / 0.2%, 2.2% / 0.22%, 2.5% / 0.3%, 2.5% / 0.25%, 4% / 0.4%, 5% / 0.5%, 6% / 0.6%. In a specific embodiment, the feed concentration is (2.2%-2.5%) / (0.22%-0.25%).

[0011] Preferably, the feed medium includes multiple feedings, for example, feedings can be performed on the 3rd, 5th, 7th, 9th, 11th and 13th days respectively.

[0012] Preferably, the culture method further comprises sugar supplementation, wherein the sugar supplementation comprises supplementation when glucose is <3 g / L, and the sugar is supplemented to a final concentration of 4 to 7 g / L, such as 4, 5, 6, or 7 g / L.

[0013] More preferably, the sugar supplementation starts on the 3rd day.

[0014] Preferably, the cell seeding density in the culture method is (0.5-1.5)×10 6 vc / mL, can be any data or range within the above range, such as 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5×10 6 vc / mL, in a specific embodiment, the inoculation density is (1.0±0.3)×10 6 vc / mL.

[0015] Preferably, the initial culture temperature of the culture method is (36±2)°C, such as 34, 35, 36, 37, or 38°C.

[0016] Preferably, the culture method includes cooling the cells in the middle and late stages of cell growth.

[0017] More preferably, the cooling culture comprises culturing at a temperature lowered to 31-35°C, such as 31, 32, 33, 34, 35°C. In a specific embodiment, the cooling culture temperature is 31 or 33°C.

[0018] Preferably, the cooling culture comprises cooling the culture to 33°C on the 5th day.

[0019] Preferably, in the culture method, the pH is 6.60-7.20, such as 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2. In a specific embodiment, the pH is 6.9±0.2.

[0020] Preferably, the culture method further comprises harvesting the supernatant after the culture.

[0021] More preferably, the harvesting step is performed when the cell viability is less than 90%, preferably when the cell viability is less than 85%, less than 80%. Further preferably, the harvesting is performed on day 9-15 of cell culture, for example, day 9, 10, 11, 12, 13, 14, 15. In a specific embodiment, the harvesting is performed on day 14.

[0022] Preferably, the culture method further comprises purifying the supernatant to obtain the Pre-F protein.

[0023] More preferably, the cell comprises an expression vector comprising a nucleic acid encoding a Pre-F protein.

[0024] Further preferably, the Pre-F protein is a fusion protein including the Pre-F protein. Further preferably, the fusion protein includes the Pre-F protein and ferritin or a mutant fragment thereof.

[0025] Preferably, the culture method comprises introducing the nucleic acid encoding the above fusion protein into an expression vector, and transfecting the expression vector into cells.

[0026] More preferably, the expression vector is a eukaryotic expression vector.

[0027] In some embodiments, the eukaryotic expression vector is an adenoviral vector.

[0028] In some embodiments, the eukaryotic expression vector is a vector expressed in 293 cells or CHO cells.

[0029] Preferably, the cells are derived from mammalian cells.

[0030] In some embodiments, the mammalian cell is selected from CHO, HEK293, SP2 / 0, BHK, C127, etc.

[0031] In some embodiments, the eukaryotic cell is a CHO cell, such as a CHO-K1Q cell.

[0032] The second aspect of the present invention provides a supernatant or Pre-F protein obtained by the above-mentioned culture method.

[0033] The supernatant includes Pre-F protein.

[0034] The third aspect of the present invention provides a use of the above-mentioned supernatant or Pre-F protein in preparing a vaccine, wherein the vaccine is used to prevent and / or treat RSV-related diseases or conditions.

[0035] A fourth aspect of the present invention provides a vaccine, which comprises the supernatant or Pre-F protein obtained by the above-mentioned culture method.

[0036] Preferably, the vaccine further comprises one or more adjuvants.

[0037] In a fifth aspect, the present invention provides a method for treating or preventing a disease or condition, the method comprising administering any of the above vaccines to a subject, wherein the disease or condition comprises a disease or condition caused by RSV.

[0038] As used herein and unless otherwise specified, the term "about" or "approximately" means within plus or minus 10% of a given value or range. Where an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.

[0039] As used herein and unless otherwise specified, the terms "comprising", "including", "having", "containing", including grammatical equivalents thereof, should generally be understood as open and non-limiting, for example, not excluding other unlisted elements or steps.

[0040] As used herein, the term "fusion protein" refers to a natural or synthetic molecule composed of one or more molecules, in which two or more peptide- or protein-based (including glycoprotein) molecules with different specificities are optionally fused together through chemical or amino acid-based linker molecules. The connection can be achieved by CN fusion or NC fusion (in the 5'→3' direction), preferably CN fusion.

[0041] As used herein, the term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including a native sequence Fc region and a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as extending from position Cys226, or from the amino acid residue at Pro230 to the carboxyl end of the heavy chain. The C-terminal lysine (residue 447 according to the EU numbering system) in the Fc region can be removed, for example during the production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody. Therefore, the composition of the complete antibody can include an antibody population from which all K447 residues have been removed, an antibody population from which no K447 residues have been removed, and an antibody population with a mixture of antibodies with and without the K447 residue.

[0042] As used herein, the term "disease" or "condition" refers to a state of life or health of a patient or individual that can be treated with the fusion proteins, pharmaceutical compositions or methods provided herein.

[0043] The term "vaccine" is a purified antigen vaccine or immunogenic composition, a subunit vaccine or immunogenic composition, an inactivated whole virus vaccine or immunogenic composition, or an attenuated virus vaccine or immunogenic composition. In some embodiments, the vaccine or immunogenic composition is a purified fusion protein.

[0044] As used herein, the term "treating" or "treatment" refers to any indicator of successful treatment or improvement of an injury, disease, pathology, or condition, including any objective or subjective parameter, such as elimination; relief; alleviation of symptoms or making the injury, pathology, or condition more tolerable to the patient; slowing the rate of degeneration or decline; or making the end point of degeneration less declining; improving the patient's physical or mental health. The treatment or improvement of symptoms can be based on objective or subjective parameters; including the results of a physical examination, neuropsychiatric examination, and / or psychiatric assessment. The term "treatment" and its conjugations may include preventing an injury, pathology, condition, or disease. In an embodiment, treatment is prevention. In an embodiment, treatment does not include prevention.

[0045] As used herein (and as is well understood in the art), "treating" or "treatment" also broadly encompasses any method for obtaining a beneficial or desired result (including a clinical result) in a subject's condition. Beneficial or desired clinical results may include, but are not limited to, alleviation or improvement of one or more symptoms or symptoms, alleviation of the extent of the disease, stabilization (i.e., non-exacerbation) of the disease state, prevention of disease transmission or spread, delay or slowing of disease progression, improvement or alleviation of the disease state, reduction of disease recurrence, and alleviation (whether partial or total, and whether detectable or undetectable). In other words, as used herein, "treatment" encompasses any cure, improvement, or prevention of a disease. Treatment may prevent the occurrence of a disease; inhibit the spread of a disease; alleviate the symptoms of a disease, completely or partially remove the underlying cause of a disease, shorten the duration of a disease, or a combination of these things.

[0046] As used herein, "patient" or "subject in need" refers to a living organism that suffers from or is susceptible to a disease or condition that can be treated by administering a pharmaceutical composition as provided herein. Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goats, sheep, cattle, deer, and other non-mammals. In some embodiments, the patient is a human.

[0047] As used herein, the term "administer" means oral administration to a subject, administration in the form of a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or implantation of a slow release device (e.g., a mini-osmotic pump). Administered by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palate, gums, nose, vagina, rectum or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular and intracranial administration. Other modes of delivery include, but are not limited to, use of liposome formulations, intravenous infusions, transdermal patches, etc. In an embodiment, administration does not include administration of any activating agent other than the narrated activating agent.

[0048] The present invention has the following beneficial effects:

[0049] 1. The present invention screens out a suitable basal medium feed medium. The protein quality obtained by using the basal medium feed medium of the present invention is relatively good, and in the preferred solution, the SEC can be higher than 90%;

[0050] 2. The present invention can ensure the stability of each batch of culture by further adjusting the process conditions such as inoculation density, feed ratio, and cooling culture. Finally, the culture process is confirmed in a 3L reactor. In the preferred scheme, the protein expression amount can reach up to 2.11g, and SEC can basically be maintained at more than 90%, and the protein quality is good and stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Cell growth curve for culture medium screening

[0052] Figure 2 Developing cell growth curves for shake flask processes

[0053] Figure 3 Analysis of DOE experimental results

[0054] Figure 4 Validation of cell growth curve for 3L reactor cell culture process. DETAILED DESCRIPTION

[0055] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are the terms and routine procedures widely used in the corresponding fields.

[0056] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0057] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0058] Among them, the following products come from:

[0059] The cells were CHO-K1, purchased from Beijing Zhaoyan Derivative Technology Co., Ltd.;

[0060] Culture medium CD04, Feed 04A, and Feed B03 were purchased from Kangsheng Biopharmaceutical Co., Ltd.;

[0061] Culture media B600S, B602S, F600aS, F600bS, and F601aS were purchased from Biogen Biotechnology Co., Ltd.

[0062] Culture media AltairCHO Medium (AltC), AltairCHO Feed, and CDFS36 were purchased from Shanghai Aupumai Biotechnology Co., Ltd.;

[0063] The culture media CHO MaxX (MaxC), MaxFA, MaxFB, and MaxFX were purchased from Shanghai Maibang Biotechnology Co., Ltd.;

[0064] The culture medium ZY-S001 was purchased from Beijing Zhaoyan Derivative Technology Co., Ltd.;

[0065] Example 1. Cell culture process development

[0066] The host cell is a CHO-K1 cell, and the Pre-F fusion protein is a fusion protein comprising Pre-F (RSV Pre-F) of respiratory syncytial virus (RSV) and Helicobacter pylori ferritin (for details, please refer to the company's existing patent, patent number: 202211654193.3, which is a part of this application and is cited as a whole in this application. For example, the fusion protein refers to SEQ ID NO.15RSV-PreF-D-NP in the document, and the nucleic acid encoding the fusion protein refers to SEQ ID NO.23RSV-PreF-D-NP in the document).

[0067] Cell culture process development is based on analysis of host cell origin, protein structure, properties and mechanism of action, and evaluates the effects of cell culture process parameters on quality attributes such as protein activity, aggregates and fragments, charge isomers, and major process attributes such as protein yield, cell growth density, cell harvest viability, etc. The main process parameters are determined and targeted research is conducted to ensure the consistency of product quality attributes and major process attributes under fermentation process conditions, and to establish a cell culture process that is efficient, robust and meets the requirements.

[0068] 1.1 Confirmation of production culture medium

[0069] The culture medium provides the necessary substances and living environment for biochemical reactions such as new cell synthesis and cell metabolism, and is the energy source for cell growth and target protein expression. Changes in the culture medium will lead to changes in cell growth and target protein expression and quality. A culture medium suitable for cell growth and expression and with protein quality and delivery time that meet project requirements is selected as the production culture medium.

[0070] The ZYB0068 project uses commercial culture media that do not contain animal or plant ingredients and have limited chemical composition for screening. Including basal culture media (CD04, B600S, B602S, AltairCHO Medium (AltC) and CHO MaxX (MaxC)) and feed culture media combinations (Feed 04A / Feed B03, F600aS / F600bS, F600aS / ZY-S001, F601aS / F600bS, AltairCHO Feed / CDFS36, MaxFA / FB, MaxFX / FB); the culture media used can support high-density cell culture, and have the advantages of increasing production and shortening the development time of cell culture processes. Because all components of the culture medium are clear, the adverse effects on cell growth and expression caused by differences between culture medium batches are avoided, which is beneficial to the stability of cell growth and expression between protein production batches and ensures product quality. The culture medium screening parameter settings are shown in Table 1: Culture medium screening process parameter settings.

[0071] Table 1: Culture medium screening process parameter settings

[0072]

[0073]

[0074] Comparing the screening results of different basal medium and feed medium combinations, the cell growth and protein expression of SF4, SF8, SF9 and SF12 combination medium were relatively good. The results are shown in Figure 1 and Table 2, Figure 1 VCD is the viable cell density, the unit is 10 6 vc / ml, represents the cell growth density, and Viability represents the cell viability. The protein expression in Table 2 is reflected by Titer (g / L).

[0075] Table 2: Protein yield results of each culture

[0076]

[0077] The basal culture medium was B602S, B600S, AltC, and the feed medium was F600aS / F600bS, F600aS / ZY-S001, F601aS / F600bS or AltairCHO Feed / CDFS36. Further, according to the protein expression results, SF4 / SF8 / SF9 / SF12 was selected for one-step purification and then protein quality detection (SEC / R-SDS-PAGE / NR-SDS-PAGE), and the results are shown in Table 3.

[0078] Table 3: Protein quality results of some culture tissues

[0079]

[0080]

[0081] The data showed that the protein quality of SP8 and SP9 was acceptable, and the protein quality of SF4 and SF12 was relatively good. Based on a comprehensive evaluation of cell growth, cell viability at harvest, protein expression, and quality of the expressed product, SF12 combination culture medium was selected for shake flask and reactor process development.

[0082] Example 2: Optimization of inoculation density and feeding strategy

[0083] During the cell culture process, the inoculation density will affect the cell growth density, integral viable cell density (IVCD) and culture time during the entire culture process, and thus affect the protein expression level. The inoculation density needs to be effectively controlled. The addition of feed provides more nutrients for cell growth and maintains cell metabolism. Cells can maintain high-density growth for a long time, thereby increasing IVCD and increasing protein expression. Too high or too low feed amounts will affect cell growth and expression, and will also have a certain impact on protein quality. In order to obtain a high-yield and stable quality cell culture process, it is necessary to optimize the cell inoculation density and feeding strategy to obtain the optimal parameters and operating space. The ZYB0068 project adopted the Fed-Batch culture mode to study the inoculation density and feed amount in a shake flask (2 factors, 3 levels). The experimental design is shown in Table 4.

[0084] Table 4: Experimental design table for shake flask process development

[0085]

[0086] The results showed that the higher the cell seeding density, the faster the cells grew in the logarithmic growth phase, and the higher the cell density during the culture process, the earlier the cells entered the stationary phase ( Figure 2 ); DOE software analysis was performed based on the protein yield and quality results (Table 5). The protein yield increased with the increase of inoculation density to 0.8×10 6 The yield was the highest when the concentration of 10 vc / ml was 0.4-1.2×10 6 The range of vc / ml is 0.8×10 6 vc / ml is optimal, and the total feed ratio is tentatively set at 25% / 2.5%. The feed ratio will be further optimized and confirmed in subsequent reactors.

[0087] Table 5: Shake flask process development yield and quality results

[0088]

[0089] Example 3 Study on Control of Culture pH and Culture Temperature

[0090] Bioreactor culture process parameters include control parameters such as pH, osmotic pressure, temperature, stirring speed and ventilation, and their effects on cell growth, protein expression and protein quality properties need to be considered comprehensively. pH is one of the important process parameters for cell culture, because it has a certain regulatory effect on the activity of intracellular enzymes, thereby affecting cell growth, expression and product quality such as molecular size isomers, charge isomers, etc. In general, cells can grow and express normally within the pH range of 6.80-7.20. In order to produce products of consistent quality, the operating range of pH needs to be studied.

[0091] Cell culture is generally carried out in a variable temperature culture method. The temperature of the cell exponential growth phase is generally higher than the culture temperature in the middle and late stages. The purpose is to increase the cell growth rate in the growth phase and obtain a sufficient number of cells to provide a basis for later protein expression. The middle and late stages of cell growth are generally the protein expression period. Lowering the culture temperature, controlling the cell growth rate, and extending the cell culture cycle are conducive to increasing the amount of protein expression. The optimization of the culture temperature also has a certain degree of influence on the purity of the target protein, protein glycosylation, and the modification of charge isomers. Given that the optimum temperature for the cell growth phase is 37°C, no optimization is required, so only the temperature parameters and cooling time in the middle and late stages of growth are studied.

[0092] The osmotic pressure of the culture medium generally affects cell growth, protein expression and product quality, such as affecting the glycosylation modification and aggregation level of the protein. The protein aggregates of the product expressed in this project are relatively high. By studying the effect of the osmotic pressure of the culture medium on the aggregates, the level of aggregates can be reduced and the yield of the product can be improved.

[0093] Different harvesting times will have a certain impact on protein yield and quality. It is necessary to study the time of culture harvesting to determine the harvesting time that meets the project quality requirements.

[0094] The ZYB0068 project adopted Fed-Batch culture. Based on the expression level and product quality requirements, the pH, temperature, basal culture medium osmotic pressure, feeding process, and culture time were studied in a 3L reactor to determine the optimal reactor culture process and culture time. The experimental design is shown in Table 5.

[0095] Table 6: Experimental design table for reactor process development

[0096]

[0097] The experimental results are shown in Table 7.

[0098] Constant temperature culture at 37°C (R14) is not conducive to maintaining cell density and viability, and the protein yield is the lowest;

[0099] With a higher cooling temperature (from 37°C to 35°C, R08) and a lower feed volume content (total feed ratio of 16.5%, R15), the cell density and viability decreased rapidly in the later stage of culture, and the protein yield was relatively low.

[0100] When the feed volume content increased (such as R04, R06, R15-17), the protein yield gradually increased, and the protein SEC purity was basically unaffected.

[0101] Osmotic pressure and pH have little effect on cell growth, metabolism, protein production and quality.

[0102] As the culture time increases, the expression level gradually increases. According to the results of cell growth, protein expression and process simplicity, 3L-R15 / R16 / R17 harvest fluid was selected for anion chromatography and composite chromatography purification. The results showed that the SEC main peak purity of proteins expressed by different processes was basically the same, and the protein SEC main peak after composite chromatography was higher, between 92.4% and 93.1%. The protein yield and quality met the requirements until the 14th day of culture.

[0103] Based on the results of cell growth, protein yield and quality, the R04, R06 and R15-17 reactor processes all have good cell growth, protein yield and quality.

[0104] Osmotic pressure has little effect on cell growth, metabolism, protein production and quality, and can fluctuate around the basal osmotic pressure, for example, 316-340 mOsm / kg.

[0105] pH has little effect on cell growth, metabolism, protein production and quality, and can fluctuate between 6.6 and 7.2.

[0106] The initial culture temperature was 36.5°C and the culture was continued until the fifth day or the cell density reached 16×10 6 vc / ml to 31-35°C, and starting from the 3rd day, add 2.0-2.5% of AltairCHO Feed and 0.20-0.25% of CDFS36 of the culture volume of the day every day (no feed is added on the day of harvest), wherein the ratio of A solution to B solution in the feed is preferably 10:1, and the cells are harvested on the 14th day or when the cell viability drops to 80% or below.

[0107] The R17 reactor process was the best process, that is, the osmotic pressure was the osmotic pressure of the basic medium, the pH was 6.9±0.2, the initial culture temperature was 36.5℃, and the culture was continued until the 5th day or the cell density reached 16×10 6vc / ml was cooled to 33°C, and starting from the 3rd day, AltairCHO Feed / CDFS36 was fed daily (no feed was added on the day of harvest), and the culture was continued until the 14th day or when the cell viability dropped to 80% or below, and then harvested.

[0108] Table 7: Protein yield and quality results for 3L reactor process development

[0109]

[0110]

[0111] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A cell culture method, characterized in that: The cells express Pre-F protein of respiratory syncytial virus, and the cell culture method comprises culturing the cells using B602S, B600S or AltC as a basic culture medium. Preferably, the basic culture medium comprises AltC.

2. The culture method according to claim 1, characterized in that The culture method includes using F600aS / F600bS, F600aS / ZY-S001, F601aS / F600bS or AltairCHO Feed / CDFS36 as feed medium. Preferably, the feed medium includes AltairCHO Feed / CDFS36. More preferably, the feed volume ratio of AltairCHO Feed / CDFS36 of the feed medium is (8-12) / 1.

3. The culture method according to claim 1 or 2, characterized in that The culture method further comprises sugar supplementation, wherein the sugar supplementation comprises supplementation when glucose is less than 3 g / L, and the sugar is supplemented to a final concentration of 4 to 7 g / L.

4. The culture method according to any one of claims 1 to 3, characterized in that: The cell seeding density in the culture method is (0.5-1.5)×10 6 vc / mL, preferably, the cell comprises an expression vector, the expression vector comprises the Pre-F protein of respiratory syncytial virus, more preferably, the Pre-F protein is a fusion protein comprising the Pre-F protein, further preferably, the cell is a mammalian cell.

5. The culture method according to any one of claims 1 to 4, characterized in that: The initial culture temperature of the culture method is (36±2)°C. Preferably, the culture method includes cooling the culture in the middle and late stages of cell growth. More preferably, the cooling culture includes lowering the temperature to 31-35°C for culture. Further preferably, in the culture method, the pH is 6.60-7.

20.

6. The culture method according to any one of claims 1 to 5, characterized in that: The culture method further comprises harvesting the supernatant. Preferably, the harvesting step is performed when the cell viability is less than 90%. More preferably, the harvesting is performed on the 9th to 16th day of cell culture.

7. The supernatant obtained by the culture method according to any one of claims 1 to 6, characterized in that: The supernatant includes Pre-F protein.

8. The supernatant according to claim 7, characterized in that The Pre-F protein is a fusion protein including the Pre-F protein.

9. Use of the supernatant according to claim 7 or 8 in preparing a vaccine, characterized in that: The vaccine is useful for preventing and / or treating RSV-associated diseases or conditions.

10. A vaccine, characterized in that The vaccine comprises the supernatant according to any one of claims 7-8.

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